Progress in FY2019 was in the following area: EFFECTS OF BEVIRIMAT ON STRUCTURE AND DYNAMICS OF THE CA-SP1 JUNCTION HELICES IN HIV-1 VIRUS-LIKE PARTICLES: Immature HIV-1 contains a protein lattice on the inner leaflet of the viral membrane, formed by the Gag polyprotein. Maturation involves cleavage of Gag into its component protein subunits by the HIV-1 protease (PR), releasing capsid protein (CA) molecules that subsequently reassemble into the mature protein lattice that encloses the viral RNA. The last cleavage step occurs between the C-terminus of CA and the N-terminus of spacer peptide 1 (SP1). This cleavage step can be inhibited by maturation inhibitor drugs such as bevirimat (BVM). It is believed that BVM binds within six-helix bundles on the inner surface of the immature Gag protein lattice, with helices formed by a segment that spans the CA-SP1 junction. However, the binding site and the effects of BVM on structure and dynamics of these junction helices are unclear. Using protocols that were developed in FY2018, we have prepared a segmentally isotopically labeled HIV-1 Gag polyprotein construct (deltaMA-CA-SP1-NC, 348 amino acids total) in which the isotopically labeled segment begins near the end of the final alpha-helix of the CA subunit (helix 11 of CA) and extends throught the NC subunit. We have prepared virus-like particles (VLPs) from this segmentally labeled protein, both with and without BVM. We have recorded 2D and 3D solid state NMR spectra of the VLPs in order to observe effects on structure and dynamics. These spectra show well-resolved signals from the C-terminus of CA and from SP1, from which we have obtained chemical shift assignments for a 17-residue segment that spans the CA-SP1 junction. Effects of BVM are very subtle, with changes in relative signal intensities but few significant changes in solid state NMR chemical shifts. We have also performed measurements of transverse 15N spin relaxation rates, which we interpret in terms of backbone motions. The relaxation rate measurements indicate a rigidification of the six-helix bundles by interaction with BVM, corresponding to a small reduction in the amplitude of backbone motion and a reduction in the correlation time of this motion. BVM does not increase the length of junction helices. To confirm that our samples do contain BVM, we have performed direct measurements of cleavage by PR and conversion of VLPs to tubular CA assemblies by electron microscopy. We find that BVM strongly inhibits this conversion process. Thus, BVM exerts a strong protective action without strongly altering the junction helix structure. A manuscript describing these experiments is currently in preparation and will be submitted at the end of FY2019. We plan to perform solid state NMR measurements on these VLPs, as soon as VLP production is scaled up to the 10 milligram level. The solid state NMR measurements will provide a more detailed structural picture of the CA-SP1 junction helix bundle, which is now known to stabilize the Gag lattice of immature HIV-1, than is currently available from our previous solid state NMR measurements on fully labeled VLPs (Bayro et al., JACS 2016) and from cryoEM measurements by the Briggs group (Schur et al., Science 2016).
